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Many clinical and preclinical studies report higher prevalence and severity of chronic pain in females. We used hyperalgesic priming with interleukin 6 (IL-6) priming and PGE(2) as a second stimulus as a model for pain chronicity. Intraplantar IL-6 induced hypersensitivity was similar in magnitude and duration in both males and females, while both paw and intrathecal PGE(2) hypersensitivity was more persistent in females. This difference in PGE(2) response was dependent on both circulating estrogen and translation regulation signaling in the spinal cord. In males, the duration of hypersensitivity was regulated by testosterone. Since the prolactin receptor (Prlr) is regulated by reproductive hormones and is female-selectively activated in sensory neurons, we evaluated whether Prlr signaling contributes to hyperalgesic priming. Using ΔPRL, a competitive Prlr antagonist, and a mouse line with ablated Prlr in the Nav1.8 sensory neuronal population, we show that Prlr in sensory neurons is necessary for the development of hyperalgesic priming in female, but not male, mice. Overall, sex-specific mechanisms in the initiation and maintenance of chronic pain are regulated by the neuroendocrine system and, specifically, sensory neuronal Prlr signaling. SIGNIFICANCE STATEMENT Females are more likely to experience chronic pain than males, but the mechanisms that underlie this sex difference are not completely understood. Here, we demonstrate that the duration of mechanical hypersensitivity is dependent on circulating sex hormones in mice, where estrogen caused an extension of sensitivity and testosterone was responsible for a decrease in the duration of the hyperalgesic priming model of chronic pain. Additionally, we demonstrated that prolactin receptor expression in Nav1.8(+) neurons was necessary for hyperalgesic priming in female, but not male, mice. Our work demonstrates a female-specific mechanism for the promotion of chronic pain involving the neuroendrocrine system and mediated by sensory neuronal prolactin receptor.

Summary Many clinical and preclinical studies report an increased prevalence and severity of chronic pain among females. Here, we identify a sex-hormone-controlled target and mechanism that regulates dimorphic pain responses. Prolactin (PRL), which is involved in many physiologic functions, induces female-specific hyperalgesia. A PRL receptor (Prlr) antagonist in the hind paw or spinal cord substantially reduced hyperalgesia in inflammatory models. This effect was mimicked by sensory neuronal ablation of Prlr. Although Prlr mRNA is expressed equally in female and male peptidergic nociceptors and central terminals, Prlr protein was found only in females and PRL-induced excitability was detected only in female DRG neurons. PRL-induced excitability was reproduced in male Prlr+ neurons after prolonged treatment with estradiol but was prevented with addition of a translation inhibitor. We propose a novel mechanism for female-selective regulation of pain responses, which is mediated by Prlr signaling in sensory neurons via sex-dependent control of Prlr mRNA translation., Graphical Abstract, Highlights • Local or spinal PRL injection induces hyperalgesia in a female-selective manner • Sensory neuron Prlr regulates tissue injury-induced pain only in females • PRL regulates excitability in Prlr+ neurons depending on sex and estrogen • Regulation of Prlr translation defines female-selective neuronal excitability, Sensory Neuroscience; Female Reproductive Endocrinology

Trigeminal (TG), dorsal root (DRG), and nodose/jugular (NG/JG) ganglia each possess specialized and distinct functions. We used RNA sequencing of two-cycle sorted Pirt-positive neurons to identify genes exclusively expressing in L3–L5 DRG, T10-L1 DRG, NG/JG, and TG mouse ganglion neurons. Transcription factor Phox2b and Efcab6 are specifically expressed in NG/JG while Hoxa7 is exclusively present in both T10-L1 and L3–L5 DRG neurons. Cyp2f2, Krt18, and Ptgds, along with pituitary hormone prolactin (Prl), growth hormone (Gh), and proopiomelanocortin (Pomc) encoding genes are almost exclusively in TG neurons. Immunohistochemistry confirmed selective expression of these hormones in TG neurons and dural nerves; and showed GH expression in subsets of TRPV1+ and CGRP+ TG neurons. We next examined GH roles in hypersensitivity in the spinal versus trigeminal systems. Exogenous GH produced mechanical hypersensitivity when injected intrathecally, but not intraplantarly. GH-induced thermal hypersensitivity was not detected in the spinal system. GH dose-dependently generated orofacial and headache-like periorbital mechanical hypersensitivity after administration into masseter muscle and dura, respectively. Periorbital mechanical hypersensitivity was reversed by a GH receptor antagonist, pegvisomant. Overall, pituitary hormone genes are selective for TG versus other ganglia somatotypes; and GH has distinctive functional significance in the trigeminal versus spinal systems.

Many clinical and preclinical studies report higher prevalence and severity of chronic pain in females. We used hyperalgesic priming with interleukin 6 (IL-6) priming and PGE2 as a second stimulus as a model for pain chronicity. Intraplantar IL-6 induced hypersensitivity was similar in magnitude and duration in both males and females, while both paw and intrathecal PGE2 hypersensitivity was more persistent in females. This difference in PGE2 response was dependent on both circulating estrogen and translation regulation signaling in the spinal cord. In males, the duration of hypersensitivity was regulated by testosterone. Since the prolactin receptor (Prlr) is regulated by reproductive hormones and is female-selectively activated in sensory neurons, we evaluated whether Prlr signaling contributes to hyperalgesic priming. Using ΔPRL, a competitive Prlr antagonist, and a mouse line with ablated Prlr in the Nav1.8 sensory neuronal population, we show that Prlr in sensory neurons is necessary for the development of hyperalgesic priming in female but not male mice. Overall, sex-specific mechanisms in the initiation and maintenance of chronic pain are regulated by the neuroendocrine system and, specifically, sensory neuronal Prlr signaling.Significance StatementFemales are more likely to experience chronic pain than males, but the mechanisms that underlie this sex difference are not completely understood. Here, we demonstrate that the duration of mechanical hypersensitivity is dependent on circulating sex hormones in mice – where estrogen caused an extension of sensitivity and testosterone was responsible for a decrease in the duration of the hyperalgesic priming model of chronic pain. Additionally, we demonstrated that Prolactin receptor expression in Nav1.8+ neurons was necessary for hyperalgesic priming in female, but not male mice. Our work demonstrates a female-specific mechanism for the promotion of chronic pain involving the neuroendrocrine system and mediated by sensory neuronal prolactin receptor.

Clinical evidence indicates brain serotonin (5-HT) stores and neurotransmission may be inadequate in subpopulations of individuals with autism, and this may contribute to characteristically impaired social behaviors. Findings that depletion of the 5-HT precursor tryptophan (TRP) worsens autism symptoms support this hypothesis. Yet dietetic studies show and parents report that many children with autism consume less TRP than peers. To measure the impact of dietary TRP content on social behavior, we administered either diets devoid of TRP, with standard TRP (0.2 gm%), or with 1% added TRP (1.2 gm%) overnight to three mouse strains. Of these, BTBRT+Itpr3tf/J and 129S1/SvImJ consistently exhibit low preference for social interaction relative to C57BL/6. We found that TRP depletion reduced C57BL/6 and 129S social interaction preference, while TRP enhancement improved BTBR sociability (p < 0.05; N= 8–10). Subsequent marble burying was similar regardless of grouping. After behavior tests, brain TRP levels and plasma corticosterone were higher in TRP enhanced C57BL/6 and BTBR, while 5-HT levels were reduced in all strains by TRP depletion (p

Zebrafish (Danio rerio) have an innate tendency to join shoals. Based on this, we refined visual choice tests to focus on social interaction and novelty preference. Our design follows mouse three-chamber sociability protocols, except testing is conducted under 940 Lux fluorescent lighting. Initially, we compared performance among zebrafish strains: inbred (AB) or wild-crossbred (WIK) from Zebrafish International Resource Center, to golden and short-fin from Petco stores. AB fish exhibited a preference for shoaling; they dwelled longest near transparent boxes containing zebrafish, while short fin favored blue boxes without fish. AB and golden exhibited a strong preference for social novelty, not evident in short-fin or WIK fish. Serotonin and cannabinoids shape mammalian social behavior, and equivalents of both receptor types are expressed in the zebrafish brain. We examined the effects of the cannabinoid receptor agonist WIN 55,212-2 (1 mg/l), or serotonin 5-HT(1A) receptor agonist buspirone (10 mg/l) on Petco short-fin social choice. Fish were bath exposed to test compounds for 10 min, under these conditions [(3) H]CP55,940 (4 nm) bound to brain with a concentration of 1.9-6.4 fmol/mg 5-30 min afterward. Social approach was measured 20 min after acclimation to the test arena. WIN 55,212-2 and buspirone increased dwelling near boxed zebrafish. In zebrafish whole-brain homogenates, buspirone displaced [(3) H] 8-hydroxy-N,N-dipropylaminotetralin (dissociation constant, K(D) = 16 ± 1.2 nm) with an inhibition constant (K(i) ) of 1.8 ± 1.0 nm lower than that of WAY 100,635 (K(i) ∼1000 nm). These fish social choice tests may enhance social behavior research, and are useful for studying the effects of genetic manipulations, pharmaceuticals or environmental toxins.